Methodology for observation of the kinetics of the refolding of denatured proteins into their native conformation will be developed. The decrease in the effective hydrodynamic volume will be observed using exclusion chromatography at high pressure. The length of the column, the resiliance of the matrix, and the geometry of the chromatographic assembly will be optimized to facilitate measurement of the kinetic parameters typically observed in the folding of small proteins at low temperature. A stopped-flow circular dichroism in instrument will be tested and modified to observe the formation of secondary structural elements and their assembly. A rapid scan stopped-flow absorbance and fluorescence spectrometer will also be tested to observe changes in the environments of intrinsic and extrinsic chromophores during folding. Studies of the mechanism of the folding of a protein containing the dinucleotide fold, thioredoxin, and variants thereof will be continued. The structure of a stable folding intermediate will be examined in detail. The equilibrium and kinetic parameters of the cis/trans isomerization of proline 76 will be measured. Additional intermediate forms will be searched for by multimixing protocols. The stability and folding of the individual globular domains will be examined as well as the role of their association in the folding mechanism. Variants of the protein will be constructed by site specitic mutations to perturb aspects of the folding mechanism.